We propose to study the stoichiometry, thermodynamics, and sources of the electrochemical H ion gradient generated by electron transport in mitochondria, which provides energy for membrane transport processes and possibly oxidative phosphorylation of ADP. Specifically, we shall measure the number of H ions ejected by electron transfer through each of the 3 energy-conserving sites of the respiratory chain, using sensitive electrodes and spectrophotometry. We shall also establish the number of H ion absorbed by mitochondria during synthesis of ATP. A survey will be made of these fundamental relationships in mitochondria from many different cell types. The relationship of H ion stoichiometry to the bioenergetics of electron transport and ATP synthesis will also be developed quantitatively, in relationship to the efficiency of cells in transducing energy of nutrients. We also propose to study the mechanism of energy-coupled Ca2 ion transport, both Ca2 ion influx and efflux in respiring mitochondria. We shall also examine the molecular signals that regulate Ca 2 ion influx and efflux in the intact cell and thus assist in regulating the many known Ca2 ion-dependent cell activities. We shall complete work on the identification of an inhibitor of calcification found in mitochondria. The relevance of these studies of energy-coupled H ion and Ca2 ion transport in mitochondria to a number of diseases, including cancer, thyroid disease, hypermetabolism, malignant hyperthemia, Reye's disease, and various aspects of normal and pathological calcification will be pursued.